The long-term objectives of this project are to elucidate the molecular mechanism of non-genomic estrogen signaling and create novel ligands that can either activate or block non-genomic signaling selectively over genomic estrogen signaling. Estrogen genomic signaling involves activation of the nuclear estrogen receptors (ERa and ERB) and subsequent transcriptional regulation of estrogen target genes. On the other hand, nongenomic signaling by estrogen results in rapid activation of ion channels, enzymes, and second messenger signaling cascades. Many of these non-genomic effects of estrogen occur in the mid-brain and may be linked to the problems of thermal regulation and mood instability that post-menopausal women face. The rapid kinetics and insensitivity of these responses to transcription/translation inhibitors rules out the involvement of the nuclear ERs acting by the classical genomic mechanism; however, the novel cellular mechanism of nongenomic signaling is unclear. This research plan is constructed around the hypothesis that a novel membrane associated G protein-coupled receptor (GPCR) for estrogen mediates at least some of the non-genomic hormone signaling. Support for this hypothesis comes from evidence that an estrogen-responsive GPCR rapidly mediates the inhibition of a specific potassium channel in hypothalamic neurons. Moreover, this physiologically relevant estrogen response is also elicited by a novel selective estrogen receptor modulator (SERM) that has no binding affinity for either ERa or ERB. In specific aim 1, an orphan GPCR is identified as a candidate receptor for estrogen and a stable cell line expressing this GPCR is developed. In specific aims 2 and 3, ligand binding and ligand activation assays are developed and carried out to establish and characterize the estrogen responsiveness of the GPCR. With these tools in place, novel SERMs will be developed that either activate (specific aim 4) or block activation (specific aim 5) of the estrogen-responsive GPCR. This research aims to define the molecular mechanisms and ligand-activation parameters of rapid, non-genomic estrogen signaling which could lead to much needed safe therapeutics for treating the symptoms of menopause. [unreadable] [unreadable]